PROJECT SUMMARY/ABSTRACT Metal-dependent lysine deacetylases (KDACs) are multi-functional proteins that mediate control of numerous cellular processes. KDACs regulate the behavior of other cellular proteins through control of the post- translational modification of lysine residues. Acetylation and deacetylation of cellular proteins have been directly associated with many cancers and other chronic diseases, as well as normal biological processes including metabolic regulation and organismal development. However, relatively few specific substrates or indirect targets of KDACs have been identified despite widespread acetylation of proteins. Moreover, the specific molecular features regulating the behavior of the KDACs themselves, both in terms of substrate selection and catalytic activity, are poorly understood. The lack of identified KDAC-specific molecular features is a critical limitation in the ability to efectively regulate KDACs and their associated pathways. Our overarching hypothesis for this project is that KDAC-specific molecular features controlling activity can be identified through comparisons of interactions with substrates and other small molecules. Our objectives are to identify the features in KDAC6 and KDAC8 that contribute to substrate selectivity, as well as to identify specific charge interactions with small ions that control the activity of each enzyme. We have recently established a combination of methods that allows for a more biologically relevant approach to answer these questions, and have preliminary results suggesting that specific ionic contacts are important for both substrate selection and overall enzymatic activity. Our aims are to characterize the molecular determinants of specific substrate and regulatory interactions of KDAC6 and KDAC8. We will utilize a combination of activity assays, direct binding assays, and computational modeling to identify specific interactions between each enzyme and its substrates. Particular emphasis will be placed on determining contributions from each of the three domains in KDAC6 and surface ionic interactions of KDAC8. KDAC6 and KDAC8 are established medical targets with a few identified protein substrates. However, the ability to selectively control these KDACs in a cellular context is limited, as inhibitors for these enzymes generally have substantial binding to other KDACs. Identification of key features of the enzyme interaction surface will improve rational approaches to inhibitor design to target specific KDACs. The mechanism of substrate selection will also contribute to the understanding of pathways regulated by KDACs and may aid in identifying other substrates, which will help establish the role of these enzymes in particular disease processes. The interactions of external ions with the enzymes will also identify alternate target sites for potential inhibitory or activating molecules. Finally, the results and methods can be extended to other KDACs to gain additional insight into the structure/function relationships of the KDAC family and their intracellular roles.